SUMMARY Coronaviruses (CoVs) have proven to be significant pathogens of both veterinary and medical importance since their discovery over 50 years ago and are responsible for two recent epidemics (SARS-CoV and MERS- CoV). The ability of CoVs to establish infection and to cause disease is dependent on their ability to inhibit the host innate immune response. Many questions still remain in this field, such as what viral factors function in vivo to prevent cytokine expression, and whether RNA sensors other than MDA5 can detect CoV RNA. I am interested in exploring both viral factors that block the immune response, as well as host proteins that promote the response and that mediate protection from infection. I have discovered that the conserved CoV macrodomain both suppresses the innate immune response and promotes in vivo replication to facilitate viral pathogenesis. Furthermore, our group and others have shown that the CoV macrodomain is an enzyme that removes ADP-ribose from proteins. These studies indicate that protein ADP-ribosylation is a mechanism used by the host to promote the innate immune response. However, neither the enzymes that catalyze the ADP- ribosylation (PARPs) or the targets of this modification are known. The central objectives in this proposal are to identify the factors that mediate antiviral ADP-ribosylation and enhance our understanding of how ADP-ribosylation impacts the innate immune response to counter CoV infection. In Aim 1 I will screen for the PARP(s) that impacts innate immunity during CoV infection and determine its localization. In Aim 2, I will systematically identify the sensor and specific step of the signaling pathway that is activated in the absence of the macrodomain. Utilizing the results from these two aims, I will then begin the process of identifying the direct protein target of the CoV macrodomain. Taken together, these aims will further define how ADP-ribosylation impacts innate immunity and CoV pathogenesis and will address the mechanism of how macrodomains combat cellular ADP-ribosylation, which remains a significant gap in the literature. A detailed understanding of the interplay between ADP-ribosylation and CoV macrodomains will provide significant insight into mechanisms of the host response and how viruses combat this response.